Tendons transmit force from muscles to bone, enabling movement, wfiile ligaments connect bone to bone, providing stabilization. Not only is the integrity and strength of the attachment important, but the location of the connections within the musculoskeletal system must be strictly regulated for efficient motion. Sports or age-related injuries to tendons or ligaments are extremely common, and although the injury can be slowly repaired, the original biomechanical properties of the tendon or ligament are never fully restored. Despite how essential these tissues are for mobility, we know very little about their molecular regulation during embryonic development. The goal of this project is to discover the pathways regulating tendon and ligament formation and differentiation in the embryo. To accomplish this, we will use the zebrafish, a powerful genetic and developmental vertebrate model system'. First, zebrafish craniofacial tendons are molecularly and histologically similar to mammalian tendons. Co-expression studies with muscle and cartilage markers demonstrate the presence of tendon transcripts at muscle-cartilage connection poi nts, thus defining a distinct population of tendon cells. To understand how zebrafish tendons form, this proposal will determine the role of distinct tissue types and known signaling pathways through loss of function analysis. Additionally, transgenic zebrafish will be generated to study tendon formation and morphogenesis over real developmental time, a technique that is unfeasible in other vertebrates. To identify novel pathways that regulate tendon development, over one thousand com pounds from a known bioactive compound library were screened and 20 putative hits were identified. Further work will focus on the validation and analysis of these hits and the pathways they target in tendon developm ent. Complementing this chemical screen, a forward genetic screen will be performed to discover and characterize new pathways regulating tendon development. These studies will directly address the major questions in the field of tendon developmental biology, and may ultimately, aid in the development of new therapies for tendon or ligament injury.